L-tryptophan is one of essential amino acids, which has been used as a feed additive or a raw material for medicines including injections and health foods owing to its hypnotic effect or tranquilizing effect. L-tryptophan has been produced by chemical synthesis, enzyme reaction and microorganism fermentation.
For chemical synthesis, high temperature and high pressure reaction is required and both D type and L type are included in the reaction product, which makes the purification process difficult. Enzyme reaction has problems of high price of indole and serine used as substrates and of instability of the enzyme, as shown in the patent description of Mitsui Toatsu (Korean Patent Publication No. 90-005773).
Therefore, L-tryptophan production has largely depended on direct fermentation using a microorganism. The production of L-tryptophan according to the conventional microorganism fermentation has been mostly carried out in auxotroph and mutant with control-region mutation of various microorganisms including E. coli and Corynebacterium. With the astonishing advancement of recombinant DNA techniques since 1980, metabolism pathway and its regulation mechanism have been disclosed. Since then, researchers have succeeded in the development of excellent recombinant strains using gene manipulation techniques, which brought remarkable increase in production.
Some of Korean Patents in relation to the production of tryptophan by direct fermentation using a microorganism describe respectively the production of tryptophan by using mutant strains having tryptophan analog resistance or auxotrophy (Korean Patent Publication Nos. 87-1813, 90-8251 and 92-7405) and the production of tryptophan by using recombinant strains (Korean Patent Publication Nos. 90-5772 and 91-5672). In the case of using a tryptophan analog resistant strain, it was a major object to overcome feed-back inhibition of enzymes in tryptophan biosynthesis. In the case of using a recombinant strain, cloning of genes involved in the tryptophan biosynthesis was a major object. And, the above methods scored a great success in fact. However, even though the conventional method for producing L-tryptophan using the conventional mutant E. coli has an advantage of L-tryptophan production through usage of inexpensive culture medium, it has a disadvantage of low L-tryptophan productivity. The present inventors considered that the production of L-tryptophan by fermentation of E. coli CJ285 (KCCM-10534, PCT/KR2004/003030) which was developed and retained by the company of the present inventors also has a problem of low productivity. Thus, the present inventors considered that the development of excellent mutant strain as a mother strain was important to maximize L-tryptophan productivity by recombinant DNA techniques.
On the other hand, the strain producing L-phenylalanine (KFCC 10066, Korean Patent Publication No. 1985-0001232) had been developed and retained by the company of the present inventors since aromatic amino acid (L-tryptophan, L-phenylalanine and L-tyrosine) can be synthesized on common metabolism pathway. So, the present inventors considered that the proper manipulation of the above strain by genetic engineering techniques could increase L-tryptophan productivity. The present inventors, therefore, used the strain producing L-phenylalanine (KFCC 10066, Korean Patent Publication No. 1985-0001232) as a mother strain for a recombinant E. coli strain producing L-tryptophan with high yield through loss of tryptophan auxotrophy, blocking of L-phenylalanine biosynthesis and enhancing of gene involved in tryptophan biosynthesis.